For the treatment of blood-related diseases typified by leukemia, it is extremely important to stably amplify and supply blood cells in an amount necessary for such treatment. Thus, to date, many researchers have attempted to efficiently amplify hematopoietic stem cells or hematopoietic progenitor cells.
Among blood cells, megakaryocytes are platelet progenitor cells, i.e., cells producing platelets and are known to form a proplatelet structure (cytoplasmic formation) to produce platelets and play an important role in therapeutic applications. Platelets are essential for blood coagulation (hemostasis). Accordingly, the demand for platelets is extremely high for leukemia, bone marrow transplantation, anticancer therapy, and so forth.
Platelet production has been attempted by administration of thrombopoietin (TPO) and in the way of differentiating umbilical cord blood or bone marrow cells into megakaryocytes, other than a method of collecting blood from blood donors so far. Recently, a method also has been attempted in which hematopoietic progenitor cells are amplified ex vivo to prepare platelets from such progenitor cells. For example, the present inventors have reported: a method for producing platelets in a relatively large amount efficiently from a sac-like structure enclosing hematopoietic progenitor cells, after the sac-like structure was prepared from human embryonic stem cells (ES cells) (PTL 1); a method for efficiently preparing mature megakaryocytes and platelets from induced pluripotent stem cells (iPS cells) in an in-vitro culture system (PTL 2); and so forth.
As described above, researches on the method for preparing platelets per se exogenously have been advanced. However, the function of prepared platelets is poorly stable. For this reason, it is necessary to develop a method that enables preparation of a large amount of functional platelets capable of being stored for an extended period of time.
Binding between platelets and an extracellular matrix is an important process for inducing blood coagulation (hemostasis, thrombus formation, and the like). It is believed that this process is initiated when a platelet receptor GPIb binds to von Willebrand factor (VWF) via an α-subunit (GPIbα). However, it has been reported that under a condition of around 37° C., a metalloproteinase ADAM17 (a disintegrin and metallopeptidase domain 17) sheds an extracellular region of GPIbα (release by cleavage), thereby inhibiting the association between GPIbα and VWF, and the platelets lose blood coagulating ability (NPLs 1 and 2).
Hence, it is anticipated that at least the activity of ADAM17 needs to be suppressed in order to keep the function of platelets prepared in vitro. Actually, the present inventors have reported that the function of platelets prepared in vitro can be kept by adding at an appropriate timing an inhibitor (such as GM6001) directly inhibiting a metalloproteinase activity or a p38 MAP kinase inhibitor indirectly inhibiting activation of the activity of metalloproteinase such as ADAM17 (PTL 3).
Nevertheless, inhibitors such as GM6001 inhibit the activities of not only ADAM17 but also other metalloproteinases (particularly MMP9 and MMP14 essential for hematopoietic function). Hence, when such inhibitors are utilized to prepare platelets in vitro, the inhibitors need to be added at a certain period when platelet production is observed most abundantly. Since such a complex task as getting right timing is required in cell culturing that is poor in consistency and reproducibility, the method for preparing platelets by utilizing these inhibitors has not been satisfactory yet in preparing a large amount of functional platelets and particularly in establishing a plant for platelet production system.
Furthermore, non-selective metalloproteinase inhibitors inhibit the activity of all of metalloproteinases such as membrane type metalloproteinases, secreted metalloproteinases and ADAMs. Accordingly, the in vivo use is believed to bring about a risk of various side effects (adverse influences caused by the inhibition of essential MMPs or ADAMs acting on different organs). Actually, since non-selective metalloproteinase inhibitors cause a severe side effect called musculoskeletal syndrome, the many developments of these inhibitors have been terminated (NPL 3). Moreover, it is suggested that an inhibitor with a hydroxamic acid structure, such as GM6001, should have mutagenicity (NPL 4). Thus, platelets obtained by a method using foregoing inhibitor are not satisfactory even in safety yet.
Meanwhile, at present, there is no effective method for storing platelets prepared from living donors, other than a method in which platelets are stored with agitating at 20° C. to 24° C. Accordingly, it seems effective to prepare platelets under room temperature conditions (20° C. to 24° C.) even when the method for suppressing the metalloproteinase activity of ADAM17 is utilized to keep the function of platelets. Nevertheless, no verification has been made under the room temperature condition at all so far whether or not it is possible to get umbilical cord blood or bone marrow cells differentiated into megakaryocytes, and whether or not it is possible to produce platelets from hematopoietic progenitor cells derived from ES cells or the like.
As described above, a compound practically usable as an active ingredient of a composition for maintaining a function of platelets has not been discovered yet. Hence, a method for obtaining functionally stable platelets in vitro and particularly a method suitable for mass production of highly safe platelets have not been established at present.